We have been investigating the regulation of the expression of SecA, a cytoplasmic ATPase that plays a major role in the translocation of proteins through the SecYEG complex in E. coli. The gene that encodes SecA resides immediately downstream of the gene that encodes SecM, and the two genes form a single operon. When secretion is impaired, a 17 amino acid motif (150FXXXXWIXXXXGIRAGP166) near the C-terminus of SecM induces a translation arrest. This motif is recognized inside the ribosome tunnel, but the mechanism of recognition is unknown. While single mutations in the motif impair recognition, we found that novel arrest-inducing peptides can be created through remodeling of the SecM C-terminus. We found that R163 is indispensable, but that flanking residues that vary in number, position, and side chain chemistry play an important secondary role in translation arrest. The observation that individual SecM variants show a distinct pattern of crosslinking to ribosomal proteins suggests that each peptide adopts a unique conformation inside the tunnel. Based on our results, we propose that translation arrest occurs when the peptide conformation specified by flanking residues moves R163 into a precise intra-tunnel location. Our data indicate that translation arrest results from extensive communication between SecM and the ribosome tunnel and help explain the striking diversity of arrest-inducing peptides found in bacteria, fungi and higher eukaryotes. We have also serendipitously obtained insight into the function of the ribosome tunnel through the analysis of an unusually long (55 amino acid) signal peptide associated with the E. coli EspP protein. The EspP signal peptide contains a 25 residue N-terminal extension (EspP1-25) that we showed inhibits signal peptide recognition by the signal recognition particle (SRP). We also found that the fusion of EspP1-25 to a cytoplasmic protein (MetE) causes MetE to aggregate. Two lines of evidence indicate that both of these effects are attributable to the conformation of EspP1-25 inside the ribosome tunnel. First, mutations in EspP1-25 that abolished its effects on protein targeting and protein folding altered the crosslinking of short nascent chains to ribosomal components. Second, a mutation in L22 that distorts the tunnel mimicked the effects of the EspP1-25 mutations on protein biogenesis. Our results provide evidence that the conformation of a polypeptide inside the ribosome tunnel can influence protein folding under physiological conditions.